The integration of cellular physiology is achieved in part through global regulatory networks which recognize stresses and activate appropriate responses. The heat shock response is one such network wherein hyperthermic stress induces the synthesis of the "heat shock proteins," or hsps. This response is ubiquitous among organisms, occurring in humans at temperatures characteristic of normal fevers. Although this response is well described, the basic mechanisms which operate to recognize metabolic stress and to activate transcription of the genes encoding the hsps are poorly understood. Nor is it clear what the functions of the hsps may be and how they may serve to protect cells from lethality at elevated temperatures. The experiments seek to clarify these issues using Drosophila melanogaster as an experimental organism. They include a biochemical approach in which a previously identified heat shock-specific transcription factor will be analyzed by DNA binding and compared with a cytoplasmic factor which appea;rs to be required for the induction of transcription of the hsp genes in vitro. It will be determined whether these two factors may interact. In addition to these positively acting factors, one of the heat shock proteins, hsp82, will be examined for its ability to act negatively on the regulation of hsp production. In addition, a genetic analysis of the heat shock system will be undertaken. These experiments will utilize strains of Drosophila which carry a gene fusion linking the structural gene encoding alcohol dehydrogenase (Adh) to the regulatory sequences for hsp70. In these flies, mutations affecting the regulation of hsp transcription can be identified by their effects on Adh. It is proposed to isolate several classes of mutations which result either in the inability to induce hsp transcription or in induction under inappropriate conditions. These different classes of mutations will be combined in double-mutant flies in order to determine the epistatic interactions among the mutations. The patterns of epistasis will allow the construction of a regulatory hierarchy, which places each of the mutations at a defined position in the regulatory system. The combination of this genetic analysis with the biochemical analysis of specific proteins and the analysis of those proteins in mutant strains will help to elucidate the mechanisms which regulate the heat shock response.